DESCRIPTION (provided by applicant: Membrane skeletons determine membrane curvature, shape, and stability for cell architecture, physiology, and pathology in both erythroid and nonerythroid cells. This proposal focuses on the membrane skeletons of red blood cells (RBCs) as well as megakaryocytes (MKs), the giant polyploid bone marrow cells that produce platelets. The paradigmatic RBC membrane skeleton consists of short actin filaments (F-actin) connected by long ?1,?1 spectrin tetramers, forming a 2D lattice attached to the membrane, while the MK membrane skeleton lines the internal demarcation membrane system (DMS), which provides the extensive plasma membrane reservoir essential for platelet formation and contains F-actin and ?1,?1 and ?2,?2 spectrins. This proposal focuses on a new player in the dynamics of the membrane skeleton, non-muscle myosin IIA (NMIIA). NMIIA consists of a dimer of two heavy chains (HC), each with two light chains (LC), with F-actin-binding motor domains at the ends of bipolar filaments. In humans, spontaneous and inherited mutations in the NMIIA HC gene (MYH9) result in rare autosomal dominant syndromes termed MYH9-related disorders (MYH9-RD). Patients have a mild bleeding tendency with macrothrombocytopenia and granulocyte inclusions, and may develop progressive kidney disease, sensorineural deafness, and cataracts, depending on the specific MYH9 mutation. This proposal will test the hypothesis that NMIIA bipolar filaments interact dynamically with F-actin in the membrane skeleton to generate forces controlling RBC membrane curvature and cell shape, as well as MK DMS formation and stability, organelle positioning, and platelet biogenesis. This proposal is based on strong preliminary data showing a) RBCs from MYH9-RD patients have abnormal shapes and altered NMIIA-membrane skeleton associations; b) bone marrow MKs from a Myh9-R702C knock-in mouse model have defects in DMS formation with disturbed ?2-spectrin and F-actin organization; and c) MYH9 motor domain mutations lead to increased HC phosphorylation at S1943 in both RBCs and MKs, indicative of reduced NMIIA contractility. The Aims are: 1) to investigate NMIIA assembly, contractility, and associations with the membrane skeleton in RBCs from patients with a spectrum of MYH9 mutations (motor domain, rod domain, C- terminal tail). The absence of anemia in patients, accessibility of RBCs, and well-understood membrane skeleton renders RBCs uniquely advantageous for biochemical approaches. 2) To investigate NMIIA functions in MK morphogenesis, DMS formation, membrane skeleton assembly, and platelet formation in mouse knock-in models with Myh9 mutations R702C (motor domain), D1424N or E1841K (rod domain), that accurately phenocopy MYH9-RD. 3) To investigate NMIIA function in MK morphogenesis and platelet formation in MKs from patients with MYH9-RD, using stem/progenitor cells isolated from peripheral blood and differentiated to MKs in vitro. These experiments will illuminate the fundamental biology of NMIIA in membrane skeleton dynamics and function, providing insights into the pathogenesis of macrothrombocytopenia in MYH9-RD.